Resorcinol Hydroxylase of Azoarcus anaerobius : Molybdenum Dependence, Activity, and Heterologous Expression
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Resorcinol Hydroxylase of Azoarcus anaerobius: Molybdenum Dependence, Activity, and Heterologous Expression Paula I. Darley1,2 · Jutta Hellstern1,3 · Bernhard Schink1 · Bodo Philipp1,4 Received: 2 April 2020 / Accepted: 25 August 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract The obligately anaerobic, denitrifying bacterium Azoarcus anaerobius strain LuFRes1 grows with resorcinol (1,3-dihydroxybenzene) as sole carbon and energy source. Resorcinol is oxidized to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by resorcinol hydroxylase (RH), an inducible membrane-bound enzyme. Sequence comparison places resorcinol hydroxylase into the group of anaerobic molybdopterin oxidoreductases and dimethyl sulfoxide reductase-like enzymes. In the large subunit, a molybdopterin-binding domain was predicted, and the small subunit most likely contains two [4Fe–4S] centers. Growth of molybdate-starved cells was inhibited by tungstate, and in vitro resorcinol hydroxylase activity was inhibited by arsenite and selenite that are known to inhibit molybdenum-containing enzymes. The two genes encoding resorcinol hydroxylase could be expressed in Escherichia coli but the products remained in inclusion bodies. All attempts to purify RH from A. anaerobius or to produce soluble, active RH in E. coli failed. Nevertheless, RH was produced as a C-terminally Strep-tagged protein from plasmid pSKM1 in Thauera aromatica AR1 transconjugants carrying a transposon insertion in the coding gene for the large (ΔrhL) or the small subunit (ΔrhS) of RH from cosmid R+. RH in the membrane fraction of wild-type transconjugant T. aromatica AR1/R+ showed a specific activity of 80 mU mg−1, and the specific activity of RH in the membranes of the complemented mutants was in the same range (80–95 mU mg−1). We conclude that RH of A. anaerobius is a membrane-bound molybdoenzyme consisting of two subunits which might require a further loosely bound subunit as membrane anchor.
Introduction Bacteria degrade aromatic compounds under oxic and anoxic conditions. In aerobic degradation of aromatics, molecular oxygen acts as a co-substrate in substrate activation and ring fission [1]. In the absence of oxygen, different strategies are used for degradation of aromatics. First, the broad variety of aromatic compounds is transformed into few central intermediates, namely benzoyl-CoA, hydroxyhydroquinone, phloroglucinol, and resorcinol, which are subsequently * Bodo Philipp bodo.philipp@uni‑muenster.de 1
University of Konstanz, Fachbereich Biologie, 78457 Constance, Germany
2
Present Address: Cambivac Ltd, Cambridge CB22 3AT, UK
3
Present Address: Novartis Pharma AG, Lichtstrasse 35, 4056 Basel, Switzerland
4
Present Address: Institute for Molecular Microbiology and Biotechnology, University of Münster, Corrensstr. 3, 48149 Münster, Germany
de-aromatized and further degraded [2–5]. Most anaerobic bacteria reduce the aromatic ring of the central intermediates before ring fission takes place by benzoyl-CoA reductases which are
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